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[Development of a double-antibody sandwich ELISA targeting the receptor binding domain of TcdB toxin of ST11 type of porcine origin].is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting of porcine origin, leaving hidden dangers for the prevention and control of . The aim of this study was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (Îº). Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution. The negative cutoff was 0.152, and no cross-reaction with 13 strains of non-ST11 type was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type in pig industry.
Wei Liang, Keji Quan, Qin Zhao, Yaomin Wu, Yu Mu, Sanjie Cao
2006 related Products with: [Development of a double-antibody sandwich ELISA targeting the receptor binding domain of TcdB toxin of ST11 type of porcine origin].50μl96tests96T 5 G100ug Lyophilized
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A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1.Fibrinogen-like protein 1 (FGL1), a member of the fibrinogen family, is a specific hepatocyte mitogen. Recently, it has been reported that FGL1 is the main inhibitory ligand of lymphocyte activating gene 3 (LAG3). Furthermore, the FGL1-LAG3 pathway has a synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway and is regarded as a promising immunotherapeutic target. However, swine FGL1 (sFGL1) has not been characterized and its detection method is lacking. In the study, the sFGL1 gene was amplified from the liver tissue of swine and then inserted into a prokaryotic expression vector, pQE-30. The recombinant plasmid pQE30-sFGL1 was transformed into JM109 competent cells. The recombinant sFGL1 was induced expression by isopropyl-Î²-d-thiogalactoside (IPTG) and the purified sFGL1 was used as an antigen to produce mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb). After identification, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for sensitive and specific detection of sFGL1 was developed. Swine FGL1 in samples was captured by anti-sFGL1 mAb followed by detection with anti-sFGL1 rabbit pAb and HRP-conjugated goat anti-rabbit IgG. The limit of detection of the developed sFLG1-DAS-ELISA is 35 pg/ml with recombinant sFLG1. Besides, it does not show cross-reactivity with the control protein. Then serum samples of PRRSV-negative and -positive pigs were tested with the established DAS-ELISA and calculated according to the equation of y=0.0735x+0.0737. The results showed that PRRSV infection enhanced the serum FGL1 levels significantly. Our research provides a platform for the research on the functional roles of swine FGL1.
Xin Zhang, Haipeng Zhu, Xu Zheng, Yunjie Jiao, Lulu Ning, En-Min Zhou, Yang Mu